The laboratory study of cells and groups of cells has been hampered by the inability to reproduce the cell's native environment on the benchtop. For example embryonic stem cells plated on microliter plates do not aggregate into homogeneous embryoid bodies in a controlled and reproducible manner; they grow across the entire bottom surface of the plate. Unlike cells cultured on a monolayer, aggregated cells tend to retain their in vivo morphology, and as a result, produce more signaling factors. By their nature, because aggregates are formed through cell-cell junctions, the possibility of anoikis is greatly reduced, allowing aggregates to remain viable over significantly longer periods of time. Aggregates allow cells to be packed into close proximity with each other. Thus, the ratio of cells to volume in a microcapsule is larger. In addition, aggregated cells produce more signaling factors and target proteins so that scale-up is attainable.
Currently, two primary techniques are employed to make cells aggregate. In the hanging drop technique, the liquid cell cultivation medium containing the cells is applied to a slide, which is then inverted. Inversion causes the drop of cultivation medium containing the cells to sink downward but not make contact with a solid surface. Because the cells have no solid surface onto which to adhere, they aggregate and, in the case of stem cells, form embryoid bodies as if they existed in vivo and the surface tension of the drops prevents escape of the cells from the drops. See Kelm and Fussenegger, Microscale tissue engineering using gravity-enforced cell assembly, TRENDS in Biotechnology 22: 195-202 (2004) for a review and description of the technique. The disadvantage of the hanging drop technique is that scaling up the technique has been unsuccessful due to the difficulty of handling large numbers of drops in parallel (i.e. in an array) and the small volumes necessitated. Another disadvantage is that it is difficult to replenish or change the composition of the culture medium or add new cells to the aggregates in these hanging drops. Top-loading, in which a defined volume of liquid is applied to a base from above and then turned over causing the drop to hang, has improved the method somewhat but not solved the array issue so the technique is still highly labor intensive.
In the spinner culture technique, cells are placed in cultivation media and spun or actively mixed. The appropriate speed of mixing conducive to the formation of aggregates must be experimentally determined: if it is too fast, the cells may be damaged or the aggregates may become excessively large. Further, the size of the resulting cell aggregates is uncontrollable and variable, and results rarely reproducible. Moreover, cells are subjected to significant shear forces during the mixing process. Shear forces are known to influence cell behavior and cell responses. Also cells with relatively weak intercellular adhesion may not readily form aggregates in this high shear environment.
One additional known method, limited to cells that divide and then form aggregates, is cell culture in methyl cellulose or soft agar. A suspension of cells is resuspended in methyl cellulose or molten soft agar and the cells are trapped at various random x, y and z locations within the viscous methylcellulose or gelled soft agar. The cells are suspended within these matrices and are unable to interact with neighboring cells to form aggregates. Only those cells, particularly cancerous or precancerous cells that can proliferate, will form aggregates by virtue of the fact that as they proliferate they grow into aggregates of cells. Ordered arrays of aggregates cannot be made because the cells are randomly dispersed within the viscous gel-like methyl cellulose or the gelled soft agar. The method is not generally applicable to cell aggregation; it is in essence a cell suspension technique as aggregation will not result for a wide variety of cell types and is limited to those cells that actively proliferate under these particular circumstances.
More recent work in the field is evidenced by United States Patent Publication No. 2003/0224510, which discloses a method of forming aggregates of cells by the application of pressure or centrifugal force to cell suspensions on permeable membranes or in hollow fibers. Also, Fukuda et al., Orderly Arrangement of Hepatocyte Spheroids on a Microfabricated Chip, Tissue Engineering 11: 125462 (2005), discloses a method of preparing spherical multicellular hepatocyte aggregates in polystyrene chip cavities with the application of a turning force. And Fukuda et al., Novel hepatocyte culture system developed using microfabrication and collagen/polyethylene glycol microcontact printing, Biomaterials, in press, (2005), discloses a polymethylmethacrylate (PMMA) microarray with cylindrical cavities having bottoms with a defined collagen-modified region onto which hepatocytes adhered and formed spheroids. The other regions of the cavities were modified with polyethylene glycol to create regions of non-adherence.
Hydrogels are colloids composed of a three-dimensional network of hydrophilic polymer chains crosslinked via chemical or physical bonding. The polymers are in the external or dispersion phase and water, present in at least 10% of the total weight (or volume), is in the internal or dispersed phase (superabsorbent hydrogels have water contents exceeding 95%). Upon cross-linking the polymer chains form a solid, three-dimensional, open-lattice type structure that can hold water or other liquids.
Hydrogels have found utility in a variety of applications: in contact lenses, as wound dressings, as medical devices such as venous catheters, as cartilage implants and in drug delivery. Hydrogels have been used widely in the development of biocompatible biomaterials, due to their low interfacial tension and low frictional surface by the presence of water on the surface. Tissue engineers use them as scaffolds for cell growth and differentiation. There are many types of hydrogels and most are suitable for some purposes and not suitable for other purposes. Hydrogels can be composed of alginate, gelatin, chitosan, pluronic, collagen, agarose, polysaccharides, proteins, polyphosphazenes, polyoxyethylene-polyoxypropylene block polymers, polyoxyethylene-polyoxypropylene block polymers of ethylene diamine, polyacrylic acids, polymethacrylic acids, copolymers of acrylic acid and methacrylic acid, polyvinyl acetates and alcohols and sulfonated polymers. Some are pH and temperature sensitive. (See Park et al., Synthesis and characterization of pH- and/or temperature-sensitive hydrogels, J. Applied Polymer Sci. 46: 659-71 (2003).) Others are light-sensitive, pressure-responsive, electro-sensitive or responsive to specific molecules. (See, Park et al., Environment-sensitive hydrogels for drug delivery, Advanced Drug Delivery Reviews 53: 321-39 (2001).) To our knowledge, employing hydrogels for cell aggregation has not been investigated, although United States Patent Publication No. 2005/0196452 to Boyan et al. discloses the use of hydrogels, especially polyvinyl alcohol hydrogels, as implants for tissue repair. The hydrogels of Boyan et al are surface-modified with a textured surface composed of pores or recesses having defined characteristics to promote attachment and acceptance of the implant and to provide physical stimulation of cells to enhance osteoblast differentiation and proliferation. It is stated that the size of the pores comprising the textured surface of the hydrogel can aid in promoting adhesion of one cell type over another.